This research was supported by the NIH Common Fund and Intramural Research Program of the National Institute of Arthritis, Musculoskeletal, and Skin Diseases.

1. Preparazione della matrice di membrana basale e rivestimento dei Plasticware <ol><li> Posizionare il congelato membrana basale matrice magazzino da -20 ° C su ghiaccio e scongelare una notte a 4 ° C. </li><li> Dopo lo scongelamento, pipetta 2 mg aliquote di matrice membrana basale in tubi Eppendorf pre-refrigerati. Conservare questi a -20 ° C fino al momento dell&#39;uso. </li><li> Per preparare piatti matrice rivestite membrana basale, scongelare un&#39;aliquota sul ghiaccio fino a quando l&#39;ultimo pezzo di ghiaccio nei scompare provetta Eppendorf (di solito entro ~ 2 ore). </li><li> Dopo lo scongelamento, aggiungere matrice membrana basale a 12 ml di freddo (4 ° C) DMEM / F12 per rendere membrana basale soluzione di rivestimento matrice. </li><li> Aggiungere la soluzione di matrice membrana basale alla nave cultura appropriata. Per un 6-pozzetti, dispensare 1 ml per pozzetto. Agitare la piastra per assicurarsi che la soluzione di matrice membrana basale copre completamente ogni pozzetto. </li><li> Parafilm sigillare la membrana basale matrice rivestite piatto / piatto, e incubare a room temperatura per 1 ora prima dell&#39;uso. In alternativa, le piastre matrice rivestite negozio membrana basale / piatti a 4 ° C e utilizzare entro 2 settimane di rivestimento. NOTA: Aggiungere ulteriore DMEM / F12 alla membrana basale matrice rivestite piatto / piatto per evitare l&#39;essiccazione. Prima di utilizzare 4 ° C membrana basale memorizzato matrice rivestite piatto / piatto, mettetelo in una cappa di sicurezza biologica e lasciare che raggiunga la temperatura ambiente per almeno 30 minuti. </li><li> Aspirare matrice membrana basale completamente prima della aggiunta del mezzo e cellule. </li></ol> 2. Preparazione di E8 media <ol><li> Preparare E8 terreno di coltura da scongelamento E8 supplemento notte a 4 ° C. </li><li> Rimuovere 10 ml di E8 media basale del magazzino da 500 ml e scartare. </li><li> Pipettare l&#39;intero flaconcino da 10 ml di E8 supplemento direttamente in 490 ml di E8 medio basale. Non caldo terreno completo E8 in un bagno d&#39;acqua a 37 ° C, come sbalzi di temperatura ripetuti possono degradare il bFGF nel complete E8 media. </li><li> Utilizzare mezzo completo E8 entro 2 settimane di aggiunta del supplemento. </li></ol> 3. scongelamento dei iPSCs <ol><li> Rimuovere una fiala di iPSCs congelati da azoto liquido e posto in ghiaccio secco. </li><li> Scongelare rapidamente la provetta in un bagno d&#39;acqua a 37 ° C; agitare il flacone a bagnomaria fino a quando solo un piccolo frammento di resti di ghiaccio. </li><li> Spruzzare il flaconcino con il 70% di etanolo e trasferimento in una cappa di sicurezza biologica. </li><li> Aggiungere 1 ml di temperatura ambiente E8 medio a gocce direttamente al flacone. </li><li> Con una pipetta 2 ml, trasferire la sospensione cellulare a gocce in 9 ml di E8 media in un tubo da 15 ml. Agitare il tubo frequentemente per assicurare che le cellule e medie mescolare bene rapidamente. </li><li> Centrifugare le cellule a 200 xg per 5 min. </li><li> Aspirare il surnatante e risospendere il pellet cellulare in un volume adeguato di E8 supplementato con 10 micron Y-27632. </li><li> Aggiungere le cellule ad un numero adeguato di seminterrato membrane pozzi matrix-rivestito, e posto a 37 ° C, 5% CO 2 incubatore durante la notte. Si raccomanda al piatto almeno 0,2 x 10 6 iPSC per pozzetto di un 6-pozzetti per consentire rapido recupero dopo lo scongelamento. </li></ol> 4. Manutenzione e Passaging routine di iPSCs <ol><li> Aggiorna E8 media giornaliera. </li><li> Monitorare la morfologia e la confluenza di cellule con un microscopio invertito. iPSCs di alta qualità crescono in colonie piatte con bordi distinti; singole colonie in possesso di un aspetto &quot;acciottolato-like&quot;. </li><li> Passage le cellule iPSCs quando raggiungono ~ 70% di confluenza. </li><li> Preparare una soluzione EDTA passaging aggiungendo 0,9 g NaCl e 500 microlitri 0,5 M EDTA a 500 ml DPBS. Mescolare bene per sciogliere NaCl, e filtro di vuoto per sterilizzare. Scaldare una aliquota della soluzione passaging in un bagno d&#39;acqua a 37 ° prima passaging. </li><li> Per il passaggio, aspirare speso terreno di coltura e lavare le cellule una volta con un uguale volume di pas caldiSaging soluzione. Aspirare, e pipetta abbastanza EDTA soluzione passaging per rivestire le celle (1 ml per pozzetto di un 6-pozzetti). </li><li> Mettere le cellule al microscopio invertito e osservare le colonie IPSC. La comparsa di fori all&#39;interno colonie e bordi rialzati dovrebbe diventare evidente entro 2 a 5 min. </li><li> Aspirare accuratamente la soluzione di EDTA passaging. </li><li> Con una pipetta 10 ml, a meno di 4 ml di E8 medio (se si utilizza un 6-pozzetti) ad alta pressione direttamente in ogni pozzetto da diversi passaggi. </li><li> Raccogliere le macchie IPSC, e suddiviso in un numero adeguato di pozzi in funzione del rapporto di divisione da 1: 8 a 1:12. Non over-pipetta, come disaggregazione dei grumi di cellule si provocherebbe un calo della redditività. </li><li> Porre la piastra in un incubatore, e oscillare la piastra posteriore e indietro e da lato a lato diverse volte per disperdere le cellule. </li></ol> 5. Preparazione di MEF e iPSCs per Tansfection <ol><li> 48 ore prima transfection, iPSCs passaggio a ~ 1: 6 in quattro o più pozzi di un 6-pozzetti membrana basale matrice rivestite, in modo che siano il 70% confluenti due giorni da qui. </li><li> Il giorno successivo, scongelare DR4 MEF in media MEF costituito da DMEM (glucosio) supplementato con 10% FBS e 1x MEM-NEAA. </li><li> Piatto DR4 MEF in due piatti da 10 cm a ~ 2 x 10 4 cellule / cm 2 e incubare una notte a 37 ° C. </li><li> Il giorno della trasfezione, modificare medio MEF a E8 supplementato con 10 mM Y-27632 30 min prima di eseguire trasfezione su iPSCs. </li><li> Opzionale: Se si desidera citometria a flusso di iPSCs post-trasfezione, rimuovere una piastra a matrice rivestita membrana basale tra 4 ° C e posto a temperatura ambiente. </li><li> Optional: 4 ore prima della trasfezione, completare pre-trasfezione cultura iPSC con Y-27632 alla concentrazione finale di 10 micron. </li></ol> 6. Gentle cellule dissociazione reagente Treatment unnd Transfection di iPSCs tramite un sistema di elettroporazione <ol><li> Rimuovere P3 soluzione trasfezione cellula primaria di 4 ° C e lasciare che raggiunga la temperatura ambiente per ~ 30 min. Aggiungere l&#39;intero complemento 100 microlitri della soluzione di trasfezione prima dell&#39;uso. </li><li> Warm dolce-cell reagente dissociazione in un bagno di 37 ° C dell&#39;acqua. </li><li> Ottenere AAVS1 Talens (PZT-AAVS1-L1 e PZT-AAVS1-R1) e AAVS1-CAG-EGFP donatore da -20 ° C. </li><li> Rimuovere iPSCs dal termostato e lavare una volta con DPBS. </li><li> Aggiungere 1 ml di delicata cellule dissociazione reagente per pozzetto e incubare iPSCs a 37 ° C per 5 minuti, o fino superiore al 50% delle cellule sono dissociato dal recipiente di coltura. </li><li> Pipettare le cellule su e giù per un paio di volte con una pipetta P1000 a dissociarsi tutte le rimanenti cellule dalla nave in cultura, e per rompere grumi IPSC. </li><li> Aggiungere 2 ml di E8 media ad ogni bene, e pipetta su e giù più volte con una pipetta 10 ml a Fürther disaggregare grumi di cellule in cellule singole NOTA: l&#39;efficienza Transfection declina in modo significativo se grumi di cellule non sono sufficientemente disaggregati. </li><li> Raccogliere iPSCs in un tubo da 15 ml e centrifugare a 100 xg per 3 min. </li><li> Aspirare il surnatante e risospendere le cellule in una quantità minima di E8 medie. </li><li> Contare le cellule utilizzando un emocitometro dopo l&#39;applicazione di una macchia vitali come 0,4% Trypan blu. Assicurarsi che le cellule siano sufficientemente dissociati, mentre il conteggio (1-3 cellule per &quot;macchia&quot;). </li><li> Dispensare 3 x 10 6 cellule in ciascuno dei due 15 ml conici, e far girare di nuovo a 100 xg per 3 min. NOTA: bassa centrifugazione velocità riduce lo stress cellulare e permette un facile risospensione di iPSCs prima elettroporazione. </li><li> Impostare il sistema di elettroporazione per il programma specifico di cellule-tipo per la linea di cellule staminali embrionali umane H9 (programma CB-150). </li><li> Dopo centrifugazione, aspirare il supernatante dal celpellets l. Per un pellet, aggiungere 10 mg del donatore HR come campione di controllo. Per l&#39;altra pellet aggiungere 10 mg del donatore HR, insieme a 5 mg di ogni TALEN (PZT-AAVS1-L1 e PZT-AAVS1-R1) come campione sperimentale. </li><li> Risospendere ogni pellet cellulare in 100 ml di soluzione di trasfezione cellulare primaria P3, e trasferire in una provetta. </li><li> Eseguire la trasfezione e aggiungere immediatamente 500 ml di temperatura ambiente medio E8 ad ogni provetta. </li><li> Trasferire il iPSCs gocce transfettate per un piatto di 10 cm contenente MEF DR4 preparati in fase 5.4. </li><li> Optional: Aggiungere qualche goccia di ciascun esperimento in un pozzetto di un 6-pozzetti membrana basale matrice rivestite se citometria a flusso anaylsis di efficienza di trasfezione è desiderato. </li><li> Ripetere i passaggi da 6,15-6,17 a finire entrambi i campioni. Il giorno seguente, lavare le cellule con DPBS due volte e passare terreno di coltura per NutriStem, che sembra sostenere la cultura iPSC su alimentatori meglio di E8. </li><li> TrAnsient picchi di espressione EGFP da 48 a 72 ore dopo la trasfezione; valutare l&#39;efficienza di trasfezione, se lo desideri. </li></ol> 7. puromicina Selezione iPSCs mirati <ol><li> Inizio selezione puromicina 72 ore dopo la trasfezione quando i iPSCs raggiungono il 70% di confluenza. </li><li> Per NCRM5 iPSCs, iniziare selezione puromicina a 0,5 mg / ml puromicina (1/2 la dose completa) diluito in NutriStem terreno di coltura. La concentrazione ottimale può variare puromicina 0,25-1 mg / MLL per alcune linee IPSC. </li><li> IPSCs cultura in NutriStem + 0,5 mg / ml puromicina per 3 giorni, rinfrescanti medio quotidiano. </li><li> Dopo 3 giorni, aumentare la concentrazione di puromicina a 1 ug / ml. </li><li> IPSCs Cultura inferiore a 1 mg / ml puromicina selezione per altri 7 a 8 giorni, o fino a quando le colonie distinte appaiono abbastanza grande per colonia picking. </li></ol> 8. Colony Picking ed espansione di iPSCs mirate <ol><li> Usa membrana basaleMatrice per rivestire una piastra a 96 pozzetti dispensando 50 ml di soluzione di rivestimento matrice membrana basale (2 mg matrice membrana basale diluito in 12 ml DMEM / F12) in ogni pozzetto. Conservare la piastra a 4 ° C per una notte prima dell&#39;uso. </li><li> Dopo aver lasciato la piastra a matrice rivestita membrana basale a venire a temperatura ambiente, aspirare la matrice membrana basale e versare 100 ml E8 supplementato con 10 micron Y-27632 in ogni pozzetto. </li><li> Inserire un microscopio invertito in una cappa di sicurezza biologica, e inumidire con il 70% EtOH per sterilizzare. </li><li> Estrarre una pipetta Pasteur di vetro su un becco Bunsen per ottenere uno strumento colonia picking ideale che ha un piccolo &quot;gancio&quot; sulla punta. Sterilizzare con il 70% EtOH, e mettere nella cappa ad asciugare. </li><li> Rimuovere il piatto contenente colonie IPSC mirati dal termostato, e collocarle nel cofano sotto il microscopio. </li><li> Scegli iPSCs tracciando un cerchio attorno al confine colonia con lo strumento della colonia di raccolta.</li><li> Utilizzare il &quot;gancio&quot; dell&#39;utensile colonia-picking per raschiare delicatamente e rimuovere la colonia COPSI dalla superficie della piastra. Per le colonie più grandi, disegnando una X con lo strumento della colonia di raccolta al quarto la colonia faciliterà la crescita delle cellule dopo la ri-placcatura. </li><li> Una volta grumi di cellule si staccano e liberamente fluttuante, usare una pipetta P200 impostato a ~ 30 microlitri per raccogliere i iPSCs. Cellule piastra direttamente nella piastra a 96 pozzetti. </li><li> Optional: usare una pipetta P20 impostato ~ 5 ml di raccogliere i iPSCs in un tubo eppendorf, quindi aggiungere 50 ml delicato cellule dissociazione reagente da digerire per 5 minuti a 37 ° C. Dopo la digestione, aggiungere 500 microlitri E8 medie nel tubo eppendorf per diluire la delicata cellule dissociazione reagente e centrifugare le cellule a 200 xg in una microcentrifuga da tavolo standard per 5 min. Poi Aspirare la maggior parte dei media e lasciare ~ 30 microlitri di riattivare e trasferire le cellule nella piastra a 96 pozzetti. NOTA: questoapproccio faciliterà la dissociazione del gruppo di cellule e rapida espansione della colonia raccolti. </li><li> Continuare colonia prelievo prima di aver raccolto un numero sufficiente di colonie IPSC (circa 20-30 colonie per esperimento è raccomandato). </li><li> Dopo colonia picking, posizionare la piastra a 96 pozzetti in un incubatore di notte. Refresh con terreno completo E8 il giorno seguente, e la cultura fino ~ 70% confluenti. </li><li> Cellule passaggio dal 96 pozzetti in un 24-ben, poi in un 6-bene, come descritto nei passaggi 4,5-4,10. NOTA: i volumi di soluzione EDTA e E8 medie dovrebbero essere proporzionalmente ridotti usando dei pozzi più piccoli di un 6-pozzetti. </li><li> Valutare il targeting successo giunzione PCR e Southern blot per confermare l&#39;integrazione cassette mirata e l&#39;assenza di vettori inseriti in modo casuale 16. </li></ol>

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Gli autori dichiarano di non avere interessi finanziari in competizione.

Le fasi più critiche per il successo generazione di AAVS1 esente mirati iPSCs umani sono: (1) efficiente di Talen e donatori plasmidi in iPSCs by trasfezione; (2) ottimizzare dissociazione di iPSCs nelle singole cellule prima trasfezione e placcatura densità dopo trasfezione; (3) ottimizzare dose ed il tempo di farmaco-selezione basata sulla crescita della linea COPSI; (4) dissezionare con cura e raccolta colonie mirate e trasferimento alla nuova piastra / bene. Rispetto ai metodi simili impiegati per la carta di Hock...

La possibilità di riprogrammare le cellule somatiche umane in cellule staminali pluripotenti indotte cellule simil-staminali embrionali (iPSCs) fu scoperto da Takahashi et al., Nel 2007 1. Fibroblasti dermici umani trasdotte con retrovirus che esprime quattro fattori di trascrizione (Il Yamanaka così soprannominato Fattori Oct3 / 4, Sox2, c-Myc e Klf4) hanno mostrato di essere molto simili alle cellule staminali embrionali umane (hESC) basati sulla morfologia, la proliferazione, l&#39;espressione genica, e lo stato epigenetico; cruciale, iPSCs sono anche in grado di differenziarsi in cellule di tutti e tre i foglietti embrionali 1. Il potenziale proliferativo e la differenziazione della capacità di iPSCs li rende strumenti molto interessanti; riprogrammando cellule di pazienti affetti da malattie specifiche, iPSCs possono essere utilizzati sia come sistemi modello in vitro di malattia e come potenziali terapie. Per quest&#39;ultimo scopo, diverse questioni devono essere affrontate prima che il pieno potenziale di iPSCsin un ambiente clinico può essere realizzato; il potenziale oncogeno di hESC coltivate in vitro e iPSCs, l&#39;uso di derivati ​​xenogeniche durante la riprogrammazione e la manutenzione delle cellule, e la necessità di tenere traccia di cellule trapiantate in vivo sono tutti ostacoli cruciali per l&#39;applicazione clinica delle cellule staminali pluripotenti (Inviato da Hentze et al. 2). Una soluzione ideale per la necessità di monitoraggio cellule differenziate post-trapianto comporterebbe un marcatore visivamente rilevabile che resiste tacere e variegatura indipendentemente dall&#39;applicazione. Espressione robusta e sostenuta di transgeni integrati è più facilmente realizzabile quando il DNA esogeno viene introdotto in porto sicuro loci; cioè, i siti genomici che consentono sufficienti trascrizione di un vettore integrato mentre allo stesso tempo mitigare perturbazioni di espressione in geni vicini 3. Uno di questi siti che è stato molto ben caratterizzati dalla sua scoperta è il virus integr adeno-associatosito zione 1 (AAVS1), nel primo introne della proteina fosfatasi 1 subunità 12C regolamentazione (PPP1R12C) gene. Questo locus è stato dimostrato non solo di permettere sostenuta e robusta espressione dei transgeni integrati attraverso il tempo prolungato nella cultura e nella differenziazione vitro 3, ma anche per proteggere i geni di perturbazione trascrizionale 4 circostante; entrambe le funzioni si pensa sia dovuto alla presenza di elementi endogeni isolante cromatina che fiancheggiano il sito AAVS1 5. Advances in strumenti di ingegneria del genoma sul proprio nell&#39;ultimo decennio hanno notevolmente facilitato la facilità e l&#39;efficienza con cui le manipolazioni genetiche in qualsiasi tipo di cellula possono essere raggiunti. Mentre i primi esperimenti riusciti invocati estremamente bassi livelli di endogena ricombinazione omologa (HR) con un donatore introdotte per ottenere gene targeting in CES 6,7, l&#39;uso di nucleasi sito-specifiche, quali nucleasi dita di zinco (ZFNs), che signi, significativamente indurre ricombinazione omologa attraverso la generazione di un doppio filamento rottura del DNA ha notevolmente aumentato l&#39;efficienza di tali esperimenti 8,9. La riproposizione di entrambi trascrizione attivatore come effettori (racconti) di pianta patogeno xanthomonas generi e le procariote cluster brevi ripetizioni palindromiche regolarmente intervallati (CRISPR) Sistema / Cas9 in efficienti nucleasi progettista site specific ha fatto gene targeting nelle cellule staminali pluripotenti un accessibile e metodologia possibile 10-13. Un recente ha descritto un metodo efficace per l&#39;integrazione stabile di una cassetta reporter fluorescente verde nel AAVS1 porto sicuro locus in iPSCs umani con nucleasi Tale (Talens) 14. Questi iPSCs mirati hanno mantenuto la loro fluorescenza, anche dopo la differenziazione diretta a cardiomiociti e il trapianto in un modello murino di infarto del miocardio (MI), che fornisce una forte evidenza per l&#39;utilità di talestabilmente pluripotenti fluorescente cellule staminali 14. Per ottenere colonie mirati, un metodo gene-trap è stato usato cui un splicing-accettore (SA), 2A sequenza peptidica auto-scissione colloca la puromicina N-acetil-transferasi (PAC) gene sotto il controllo del promotore PPP1R12C endogena; quindi, solo iPSCs che hanno incorporato il donatore DNA a livello del locus AAVS1 esprimono PAC, rendendoli selezionabili in base puromicina-resistenza; (Figura 1, 15). Dettagli Questo protocollo le procedure di generare AAVS1-GFP iPSCs segnalato nel recente documento 14, tra cui il processo di trasfettando iPSCs con Talens e 9,8 kb donatori di integrare un frammento di 4.2kb DNA nel locus AAVS1 porto sicuro, selezionando iPSCs basati su puromicina-resistenza, e le colonie di prelievo per l&#39;espansione clonale. Le tecniche qui descritte possono essere applicate a vari esperimenti di ingegneria genoma.

<table border="0" cellpadding="0" cellspacing="0" width="1437">
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		<tr height="21">
			<td height="21" style="height:21px;width:588px;">NAME OF MATERIAL/EQUIPMENT</td>
			<td style="width:123px;">COMPANY</td>
			<td style="width:123px;">CATALOG #</td>
			<td style="width:603px;">COMMENTS/DESCRIPTION</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Matrigel Growth Factor Reduced (GFR) Basement Membrane Matrix, *LDEV-Free, 10mL</td>
			<td>Corning</td>
			<td>354230</td>
			<td>Store at -20&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM/F-12</td>
			<td>Life Technologies</td>
			<td>11320-033</td>
			<td>Store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Costar 6 Well Clear TC-Treated Multiple Well Plates</td>
			<td>Corning</td>
			<td>3506</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Essential 8 Medium</td>
			<td>Life Technologies</td>
			<td>A1517001</td>
			<td>Store basal medium at 4&#176;C. Store supplement at -20&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Y-27632 dihydrochloride</td>
			<td>Tocris</td>
			<td>1254</td>
			<td>Store at room temp. Once dissolved in H2O, store at -20&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Chloride</td>
			<td>Sigma</td>
			<td>S5886-500G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">UltraPure 0.5M EDTA, pH 8.0</td>
			<td>Life Technologies</td>
			<td>15575-020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DPBS, no calcium, no magnesium</td>
			<td>Life Technologies</td>
			<td>14190-250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100mm TC-Treated Culture Dish</td>
			<td>Corning</td>
			<td>430167</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DR4 MEF 2M IRR - Academic</td>
			<td>GlobalStem</td>
			<td>GSC-6204G</td>
			<td>Store in liquid Nitrogen.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM, high glucose, pyruvate</td>
			<td>Life Technologies</td>
			<td>11995-040</td>
			<td>Store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Defined Fetal Bovine Serum, US Origin</td>
			<td>HyClone</td>
			<td>SH30070.03</td>
			<td>Store at -20&#176;C. Thaw at 4&#176;C overnight and aliquot. Store aliquots at -20&#176;C until needed.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MEM Non-Essential Amino Acids Solution (100X)</td>
			<td>Life Technologies</td>
			<td>11140-050</td>
			<td>Store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4D-Nucleofector Core unit&#160;</td>
			<td>Lonza</td>
			<td>AAF-1001B</td>
			<td>part of the electroporation system</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4D-Nucleofector X unit&#160;</td>
			<td>Lonza</td>
			<td>AAF-1001X</td>
			<td>part of the electroporation system</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">P3 Primary Cell 4D-Nucleofector X Kit L (24 RCT)</td>
			<td>Lonza</td>
			<td>V4XP-3024</td>
			<td>Upon arrival, remove Primary Cell Solution and supplement and store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">StemPro Accutase Cell Dissociation Reagent</td>
			<td>Life Technologies</td>
			<td>A1110501</td>
			<td>Store at -20&#176;C. Thaw overnight at 4&#176;C and warm an aliquot in a 37&#176;C water bath before use.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NutriStem XF/FF Culture Medium</td>
			<td>Stemgent</td>
			<td>01-0005</td>
			<td>Store at -20&#176;C. Thaw overnight at 4&#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AAVS1 TALENs (pZT-AAVS1-L1 and pZT-AAVS1-R1)</td>
			<td>Addgene</td>
			<td>52637 and 52638</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AAVS1-CAG-EGFP Homologous Recombination donor</td>
			<td>Addgene</td>
			<td>22212</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Puromycin Dihydrochloride</td>
			<td>Life Technologies</td>
			<td>A11138-03</td>
			<td>Store at -20&#176;C. Prepare working aliquots of 1 mg/mL in ddH2O.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Disposable Borosilicate Glass Pasteur Pipets</td>
			<td>Fisher Scientific</td>
			<td>13-678-20A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sorvall Legend XTR (Refrigerated), 120V 60Hz</td>
			<td>Thermo Scientific</td>
			<td>75-004-521</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TX-750 4 &#215; 750mL Swinging Bucket Rotor</td>
			<td>Thermo Scientific</td>
			<td>75003607</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypan Blue Solution, 0.4%</td>
			<td>Life Technologies</td>
			<td>15250-061</td>
			<td></td>
		</tr>
	</tbody>
</table>

transfection, selection, and colony-picking of human induced pluripotent stem cells talen-targeted with a gfp gene into the aavs1 safe harbor

Targeted transgene addition can provide persistent gene expression while circumventing the gene silencing and insertional mutagenesis caused by viral vector mediated random integration. This protocol describes a universal and efficient transgene targeted addition platform in human iPSCs based on utilization of validated open-source TALENs and a gene-trap-like donor to deliver transgenes into a safe harbor locus. Importantly, effective gene editing is rate-limited by the delivery efficiency of gene editing vectors. Therefore, this protocol first focuses on preparation of iPSCs for transfection to achieve high nuclear delivery efficiency. When iPSCs are dissociated into single cells using a gentle-cell dissociation reagent and transfected using an optimized program, >50% cells can be induced to take up the large gene editing vectors. Because the AAVS1 locus is located in the intron of an active gene (PPP1R12C), a splicing acceptor (SA)-linked puromycin resistant gene (PAC) was used to select targeted iPSCs while excluding random integration-only and untransfected cells. This strategy greatly increases the chance of obtaining targeted clones, and can be used in other active gene targeting experiments as well. Two weeks after puromycin selection at the dose adjusted for the specific iPSC line, clones are ready to be picked by manual dissection of large, isolated colonies into smaller pieces that are transferred to fresh medium in a smaller well for further expansion and genetic and functional screening. One can follow this protocol to readily obtain multiple GFP reporter iPSC lines that are useful for in vivo and in vitro imaging and cell isolation.

Una visualizzazione del protocollo è fornita in figura 2, con i periodi durante i quali iPSCs sono coltivate in mezzo diverso evidenziata da una verde per E8 o blu per NutriStem. E &#39;importante per trasfettare solo iPSCs di alta qualità; esaminare piatti della cultura in tutta la manutenzione ordinaria e verificare che le culture IPSC contengono colonie principalmente distinti recanti una morfologia ciottoli-simile (Figura 3A); cellule differenziate non dovrebbe occupare più del 1...

Watch this Scientific Journal Video about Transfection, Selection, and Colony-picking of Human Induced Pluripotent Stem Cells TALEN-targeted with a GFP Gene into the AAVS1 Safe Harbor at JoVE.com

Transfection, Selection, and Colony-picking of Human Induced Pluripotent Stem Cells TALEN-targeted with a GFP Gene into the AAVS1 Safe Harbor

TALEN-mediated gene editing at the safe harbor AAVS1 locus enables high-efficiency transgene addition in human iPSCs. This protocol describes the procedures for preparing iPSCs for TALEN and donor vector delivery, transfecting iPSCs, and selecting and isolating iPSC clones to achieve targeted integration of a GFP gene to generate reporter lines.

Transfection, selezione, e Colonia-picking di cellule staminali umane pluripotenti indotte TALEN-mirati con Gene GFP nel AAVS1 Safe Harbor

Targeted transgene addition can provide persistent gene expression while circumventing the gene silencing and insertional mutagenesis caused by viral ...

transfection-selection-colony-picking-human-induced-pluripotent-stem

Research

JoVE Journal

Developmental Biology

19.3K Views.  National Institutes of Health. TALEN-mediated gene editing at the safe harbor AAVS1 locus enables high-efficiency transgene addition in human iPSCs. This protocol describes the procedures for preparing iPSCs for TALEN and donor vector delivery, transfecting iPSCs, and selecting and isolating iPSC clones to achieve targeted integration of a GFP gene to generate reporter lines.

Video: Transfection, Selection, and Colony-picking of Human Induced Pluripotent Stem Cells TALEN-targeted with a GFP Gene into the AAVS1 Safe Harbor

Three-dimensional Quantification of Dendritic Spines from Pyramidal Neurons Derived from Human Induced Pluripotent Stem Cells

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are distributed along the dendrites. Their morphology is largely dependent on neuronal activity, and they are dynamic. Dendritic spines express glutamatergic receptors (AMPA and NMDA receptors) on their surface and at the levels of postsynaptic densities. Each spine allows the neuron to control its state and local activity independently. Spine morphologies have been extensively studied in glutamatergic pyramidal cells of the brain cortex, using both in vivo approaches and neuronal cultures obtained from rodent tissues. Neuropathological conditions can be associated to altered spine induction and maturation, as shown in rodent cultured neurons and one-dimensional quantitative analysis 1. The present study describes a protocol for the 3D quantitative analysis of spine morphologies using human cortical neurons derived from neural stem cells (late cortical progenitors). These cells were initially obtained from induced&#160;pluripotent stem cells. This protocol allows the analysis of spine morphologies at different culture periods, and with possible comparison between induced&#160;pluripotent stem cells obtained from control individuals with those obtained from patients with psychiatric diseases.

Dendritic spines of pyramidal neurons are the sites of most excitatory synapses in mammalian brain cortex. This method describes a 3D quantitative analysis of spine morphologies in human cortical pyramidal glutamatergic neurons derived from induced&#160;pluripotent stem cells.

Quantificazione tridimensionale di spine dendritiche da piramidale neuroni derivate da cellule staminali umane pluripotenti indotte

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are ...

Ricerca

Biologia dello sviluppo

Cultivate Primary Nasal Epithelial Cells from Children and Reprogram into Induced Pluripotent Stem Cells

Nasal epithelial cells (NECs) are the part of the airways that respond to air pollutants and are the first cells infected with respiratory viruses. They are also involved in many airway diseases through their innate immune response and interaction with immune and airway stromal cells. NECs are of particular interest for studies in children due to their accessibility during clinical visits. Human induced pluripotent stem cells (iPSCs) have been generated from multiple cell types and are a powerful tool for modeling human development and disease, as well as for their potential applications in regenerative medicine. This is the first protocol to lay out methods for successful generation of iPSCs from NECs derived from pediatric participants for research purposes. It describes how to obtain nasal epithelial cells from children, how to generate primary NEC cultures from these samples, and how to reprogram primary NECs into well-characterized iPSCs. Nasal mucosa samples are useful in epidemiological studies related to the effects of air pollution in children, and provide an important tool for studying airway disease. Primary nasal cells and iPSCs derived from them can be a tool for providing unlimited material for patient-specific research in diverse areas of airway epithelial biology, including asthma and COPD research.

This publication demonstrates methods for successful sampling and culture of nasal epithelial mucosa from children, and reprogramming these cells to induced Pluripotent Stem Cells (iPSCs).

Coltivare cellule primarie epiteliali nasali da bambini e riprogrammare in cellule staminali pluripotenti indotte

Nasal epithelial cells (NECs) are the part of the airways that respond to air pollutants and are the first cells infected with respiratory viruses. They ...

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

The placenta is the first organ to develop during embryogenesis and is required for the survival of the developing embryo. The placenta is comprised of various trophoblastic cells that differentiate from the extra-embryonic trophectoderm cells of the preimplantation blastocyst. As such, our understanding of the early differentiation events of the human placenta is limited because of ethical and legal restrictions on the isolation and manipulation of human embryogenesis. Human pluripotent stem cells (hPSCs) are a robust model system for investigating human development and can also be differentiated in vitro into trophoblastic cells that express markers of the various trophoblast cell types. Here, we present a detailed protocol for differentiating hPSCs into trophoblastic cells using bone morphogenic protein 4 and inhibitors of the Activin/Nodal signaling pathways. This protocol generates various trophoblast cell types that can be transfected with siRNAs for investigating loss-of-function phenotypes or can be infected with pathogens. Additionally, hPSCs can be genetically modified and then differentiated into trophoblast progenitors for gain-of-function analyses. This in vitro differentiation method for generating human trophoblasts starting from hPSCs overcomes the ethical and legal restrictions of working with early human embryos, and this system can be used for a variety of applications, including drug discovery and stem cell research.

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

Here, we present a protocol to efficiently generate human trophoblastic cells from human pluripotent stem cells using bone morphogenic protein 4 and inhibitors of the Activin/Nodal pathways. This method is suitable for the efficient differentiation of human pluripotent stem cells and can generate large quantities of cells for genetic manipulation.

La differenziazione in vitro di pluripotenti umane Cellule Staminali in cellule trofoblastiche

The placenta is the first organ to develop during embryogenesis and is required for the survival of the developing embryo. The placenta is comprised of ...

Protocol for the Differentiation of Human Induced Pluripotent Stem Cells into Mixed Cultures of Neurons and Glia for Neurotoxicity Testing

Human pluripotent stem cells can differentiate into various cell types that can be applied to human-based in vitro toxicity assays. One major advantage is that the reprogramming of somatic cells to produce human induced pluripotent stem cells (hiPSCs) avoids the ethical and legislative issues related to the use of human embryonic stem cells (hESCs). HiPSCs can be expanded and efficiently differentiated into different types of neuronal and glial cells, serving as test systems for toxicity testing and, in particular, for the assessment of different pathways involved in neurotoxicity. This work describes a protocol for the differentiation of hiPSCs into mixed cultures of neuronal and glial cells. The signaling pathways that are regulated and/or activated by neuronal differentiation are defined. This information is critical to the application of the cell model to the new toxicity testing paradigm, in which chemicals are assessed based on their ability to perturb biological pathways. As a proof of concept, rotenone, an inhibitor of mitochondrial respiratory complex I, was used to assess the activation of the Nrf2 signaling pathway, a key regulator of the antioxidant-response-element-(ARE)-driven cellular defense mechanism against oxidative stress.

Human induced pluripotent stem cells (hiPSCs) are considered a powerful tool for drug and chemical screening and for the development of new in vitro models for toxicity testing, including neurotoxicity. Here, a detailed protocol for the differentiation of hiPSCs into neurons and glia is described.

Protocollo per la differenziazione delle cellule staminali pluripotenti indotte dall&#39;uomo in culture miste di neuroni e glia per la sperimentazione della neurotossicità

Human pluripotent stem cells can differentiate into various cell types that can be applied to human-based in vitro toxicity assays. One major advantage is ...

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing a detailed and thorough protocol is to clearly demonstrate each step and to make this readily available to researchers in the field. This protocol results in a homogenous layer of RPE with minimal or no manual dissection needed. The method presented here has been shown to be effective for induced pluripotent stem cells (iPSC) and human embryonic stem cells. Additionally, we describe methods for cryopreservation of intermediate cell banks that allow long-term storage. RPE generated using this protocol might be useful for iPSC disease-in-a-dish modeling or clinical application.

This protocol describes how to produce retinal pigment epithelial cells (RPE) from pluripotent stem cells. The method uses a combination of growth factors and small molecules to direct the differentiation of stem cells into immature RPE in fourteen days and mature, functional RPE after three months.

Rapida, regia di differenziazione delle cellule epiteliali del pigmento retinico da cellule staminali pluripotenti umane embrionali o indotto

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...

Using Human Induced Pluripotent Stem Cell-derived Hepatocyte-like Cells for Drug Discovery

The ability to differentiate human induced pluripotent stem cells (iPSCs) into hepatocyte-like cells (HLCs) provides new opportunities to study inborn errors in hepatic metabolism. However, to provide a platform that supports the identification of small molecules that can potentially be used to treat liver disease, the procedure requires a culture format that is compatible with screening thousands of compounds. Here, we describe a protocol using completely defined culture conditions, which allow the reproducible differentiation of human iPSCs to hepatocyte-like cells in 96-well tissue culture plates. We also provide an example of using the platform to screen compounds for their ability to lower Apolipoprotein B (APOB) produced from iPSC-derived hepatocytes generated from a familial hypercholesterolemia patient. The availability of a platform that is compatible with drug discovery should allow researchers to identify novel therapeutics for diseases that affect the liver.

The protocol presented here describes a platform for identifying small molecules for the treatment of liver disease. A step-by-step description is presented detailing how to differentiate iPSCs into cells with hepatocyte characteristics in 96-well plates, and to use the cells to screen for small molecules with potential therapeutic activity.

Utilizzando umani indotti dell'epatocita-come di derivati da cellule staminali pluripotenti per scoperta della droga

The ability to differentiate human induced pluripotent stem cells (iPSCs) into hepatocyte-like cells (HLCs) provides new opportunities to study inborn ...

A Familial Hypercholesterolemia Human Liver Chimeric Mouse Model Using Induced Pluripotent Stem Cell-derived Hepatocytes

Familial hypercholesterolemia (FH) is mostly caused by low-density lipoprotein receptor (LDLR) mutations and results in an increased risk of early-onset cardiovascular disease due to marked elevation of LDL cholesterol (LDL-C) in blood. Statins are the first line of lipid-lowering drugs for treating FH and other types of hypercholesterolemia, but new approaches are emerging, in particular PCSK9 antibodies, which are now being tested in clinical trials. To explore novel therapeutic approaches for FH, either new drugs or new formulations, we need appropriate in vivo models. However, differences in the lipid metabolic profiles compared to humans are a key problem of the available animal models of FH. To address this issue, we have generated a human liver chimeric mouse model using FH induced pluripotent stem cell (iPSC)-derived hepatocytes (iHeps). We used Ldlr-/-/Rag2-/-/Il2rg-/- (LRG) mice to avoid immune rejection of transplanted human cells and to assess the effect of LDLR-deficient iHeps in an LDLR null background. Transplanted FH iHeps could repopulate 5-10% of the LRG mouse liver based on human albumin staining. Moreover, the engrafted iHeps responded to lipid-lowering drugs and recapitulated clinical observations of increased efficacy of PCSK9 antibodies compared to statins. Our human liver chimeric model could thus be useful for preclinical testing of new therapies to FH. Using the same protocol, similar human liver chimeric mice for other FH genetic variants, or mutations corresponding to other inherited liver diseases, may also be generated.

Here, we present a protocol to generate a human liver chimeric mouse model of familial hypercholesterolemia using human induced pluripotent stem cell-derived hepatocytes. This is a valuable model for testing new therapies for hypercholesterolemia.

Un modello di Mouse chimerici del fegato umano di ipercolesterolemia familiare facendo uso degli epatociti derivati da cellule staminali pluripotenti indotte

Familial hypercholesterolemia (FH) is mostly caused by low-density lipoprotein receptor (LDLR) mutations and results in an increased risk of early-onset ...

RNA-based Reprogramming of Human Primary Fibroblasts into Induced Pluripotent Stem Cells

Induced pluripotent stem cells (iPSCs) have proven to be a valuable tool to study human development and disease. Further advancing iPSCs as a regenerative therapeutic requires a safe, robust, and expedient reprogramming protocol. Here, we present a clinically relevant, step-by-step protocol for the extremely high-efficiency reprogramming of human dermal fibroblasts into iPSCs using a non-integrating approach. The core of the protocol consists of expressing pluripotency factors (SOX2, KLF4, cMYC, LIN28A, NANOG, OCT4-MyoD fusion) from in vitro transcribed messenger RNAs synthesized with modified nucleotides (modified mRNAs). The reprogramming modified mRNAs are transfected into primary fibroblasts every 48 h together with mature embryonic stem cell-specific microRNA-367/302 mimics for two weeks. The resulting iPSC colonies can then be isolated and directly expanded in feeder-free conditions. To maximize efficiency and consistency of our reprogramming protocol across fibroblast samples, we have optimized various parameters including the RNA transfection regimen, timing of transfections, culture conditions, and seeding densities. Importantly, our method generates high-quality iPSCs from most fibroblast sources, including difficult-to-reprogram diseased, aged, and/or senescent samples.

Here we describe a clinically relevant, high-efficiency, feeder-free method to reprogram human primary fibroblasts into induced pluripotent stem cells using modified mRNAs encoding reprogramming factors and mature microRNA-367/302 mimics. Also included are methods to assess reprogramming efficiency, expand clonal iPSC colonies, and confirm expression of the pluripotency marker TRA-1-60.

RNA-ha basato riprogrammazione dei fibroblasti primari umani in cellule staminali pluripotenti indotte

Induced pluripotent stem cells (iPSCs) have proven to be a valuable tool to study human development and disease. Further advancing iPSCs as a regenerative ...

Efficient Differentiation of Human Pluripotent Stem Cells into Liver Cells

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver failure&#8212;which can be caused by chronic alcohol intake, hepatitis, acute poisoning, or other insults&#8212;is a severe condition that can culminate in bleeding, jaundice, coma, and eventually death. However, approaches to treat liver failure, as well as studies of liver function and disease, have been stymied in part by the lack of a plentiful supply of human liver cells. To this end, this protocol details the efficient differentiation of human pluripotent stem cells (hPSCs) into hepatocyte-like cells, guided by a developmental roadmap that describes how liver fate is specified across six consecutive differentiation steps. By manipulating developmental signaling pathways to promote liver differentiation and to explicitly suppress the formation of unwanted cell fates, this method efficiently generates populations of human liver bud progenitors and hepatocyte-like cells by days 6 and 18 of PSC differentiation, respectively. This is achieved through the temporally-precise control of developmental signaling pathways, exerted by small molecules and growth factors in a serum-free culture medium. Differentiation in this system occurs in monolayers and yields hepatocyte-like cells that express characteristic hepatocyte enzymes and have the ability to engraft a mouse model of chronic liver failure. The ability to efficiently generate large numbers of human liver cells in vitro has ramifications for treatment of liver failure, for drug screening, and for mechanistic studies of liver disease.

This protocol details a monolayer, serum-free method to efficiently generate hepatocyte-like cells from human pluripotent stem cells (hPSCs) in 18 days. This entails six steps as hPSCs sequentially differentiate into intermediate cell-types such as the primitive streak, definitive endoderm, posterior foregut and liver bud progenitors before forming hepatocyte-like cells.

Differenziazione efficiente delle cellule staminali pluripotenti umane nelle cellule del fegato

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver ...

Generation of 3D Skin Organoid from Cord Blood-derived Induced Pluripotent Stem Cells

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily functions. Biomimetics is the imitation of the models, systems, and elements of nature for the purpose of solving complex human problems1. Skin biomimetics is a useful tool for in vitro disease research and in vivo regenerative medicine. Human induced pluripotent stem cells (iPSCs) have the characteristic of unlimited proliferation and the ability of differentiation to three germ layers. Human iPSCs are generated from various primary cells, such as blood cells, keratinocytes, fibroblasts, and more. Among them, cord blood mononuclear cells (CBMCs) have emerged as an alternative cell source from the perspective of allogeneic regenerative medicine. CBMCs are useful in regenerative medicine because human leukocyte antigen (HLA) typing is essential to the cell banking system. We provide a method for the differentiation of CBMC-iPSCs into keratinocytes and fibroblasts and for generation of a 3D skin organoid. CBMC-iPSC-derived keratinocytes and fibroblasts have characteristics similar to a primary cell line. The 3D skin organoids are generated by overlaying an epidermal layer onto a dermal layer. By transplanting this 3D skin organoid, a humanized mice model is generated. This study shows that a 3D human iPSC-derived skin organoid may be a novel, alternative tool for dermatologic research in vitro and in vivo.

We propose a protocol that shows how to differentiate induced pluripotent stem cell-derived keratinocytes and fibroblasts and generate a 3D skin organoid, using these keratinocytes and fibroblasts. This protocol contains an additional step of generating a humanized mice model. The technique presented here will improve dermatologic research.

Generazione di pelle 3D Organoid dal cordone emoderivati indotta da cellule staminali pluripotenti indotte

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily ...